Gene transfer of vasostatin, a calreticulin fragment, into neuroendocrine tumor cells results in enhanced malignant behavior

Vasostatin, a fragment of calreticulin, was transfected in the BON cell line to evaluate the feasibility of using it for gene therapy in neuroendocrine tumors. Vasostatin transfected cells were subcutaneously inoculated in nude mice. Burkitt lymphoma cell line, CA46, colorectal adenocarcinoma cell line, SW480, as well as endothelial cells PAE and SVEC4 were used for evaluating the function of vasostatin. The results demonstrated that vasostatin transfer caused enhanced malignant behavior of neuroendocrine tumor cell line, BON. Cell adhesion, spreading and cellular invasion were also enhanced in vasostatin-expressing BON cells. Tumor suppressor genes including p53, nm23, Rb and vinculin were down-regulated. Moreover, cell cycle regulatory protein, p27kip1, and cell differentiation-related protein kinase, PKR, were also significantly down-regulated. Furthermore, expression of NKG2D ligands, MICA and MICB, were down-regulated. Mice implanted with vasostatin-expressing BON cells showed an earlier and faster tumor growth compared to wild type. Anti-proliferative effects of vasostatin could not be proven in other cells except in PAE. These results indicated that vasostatin does probably not have a tumor growth inhibitory effect by itself, but rather modulates processes which are necessary for tumor growth. Therefore, one should be very careful when using vasostatin as an anti-tumoral agent in clinical trials, at least for neuroendocrine tumors.     

Establishment and characterization of a human carcinoid in nude mice and effect of various agents on tumor growth.

The authors have established a long-term tissue culture cell line (BON) derived from a metastatic human carcinoid tumor of the pancreas. The cells have been in continuous passage for 46 months. Tissue culture cells produce tumors in a dose-dependent fashion after SC inoculation of cell suspensions in athymic nude mice. BON tumors, grown in nude mice, are histologically identical to the original tumor; they possess gastrin and somatostatin receptors, synthesize serotonin and chromogranin A, and have a doubling time of approximately 13 days. The antiproliferative effects of the long-acting somatostatin analogue, SMS 201-995 (300 micrograms/kg, t.i.d.), and 2% alpha-difluoromethylornithine on BON xenografts in nude mice were examined. Tumor size was significantly decreased by day 14 of treatment with either agent and at all points of analysis thereafter until the animals were killed (day 33). In addition, tumor weight, DNA, RNA, and protein contents were significantly decreased in treated mice compared with controls. Establishment of this human carcinoid xenograft line, BON, provides an excellent model to study further the biological behavior of carcinoid tumors and the in vivo effect of chemotherapeutic agents on tumor growth.     

Transduction of human hepatocellular carcinoma cells with human alpha-interferon gene via retroviral vector

AIM:To investigate the therapeutic potential of gamma interferon (IFN-alpha) genemodified human hepatocellular carcinoma (HCC) cells.METHODS:The IFN-alpha gene was introduced retrovirally into four HCC cell lines.Secreted IFN-alpha activity was assessed using bioassay. The expression of MHC molecules was detected by FACS.Tumorigenicity was analysed by tumor formation in nude mice.RESULTS:Four IFN-alpha gene transduced HCC cell lines secreted different amounts of IFN-alpha, as in the same case of five clones derived from one HCC cell line. Transduction with IFN-alpha caused significant increase in the expression of major histocompatibility complex (MHC) antigens on HCC cells. The expression of HLA class I was increased by 2-3 times in terms of mean fluorescence intensities, while for class II expression, the percentage of positive cells augmented from < 10% to &lg 50%. When equal amount of tumor cells were injected into nude mice, the tumor igenicity some transduced cells decreased dramantically.CONCLUSION:IFN-alpha gene transduction can convert weakly imunogenic HCC cells to activate antitumor immune response, and further pave the way for the future use of such gene modified tumor cells as a modality for the cancer immunotherapy.     

Elevated expression of proprotein convertases alters breast cancer cell growth in response to estrogen and tamoxifen

Two proprotein convertase cDNAs, PC1 and furin, were stably transfected into the human breast cancer cell line MCF-7. The PC1 or furin over-expressing cells possessed an altered morphology. When grown in vitro in a serum-free medium, the population doubling time of the convertase-transfected cells was twice that of wild-type (WT) cells. High concentrations of estradiol stimulated the growth of all three cell types to a similar extent; however, at low concentrations of estradiol, the convertase-transfected cells grew more slowly than WT cells. In athymic nude mice implanted with 5 mg estradiol pellets, the growth of tumors of convertase-transfected MCF-7 cells was stimulated to a degree similar to that of WT MCF-7 tumors. However, in mice implanted with lower-dose (1.5 mg) estradiol pellets, the tumors of PC1- or furin-transfected MCF-7 cells grew approximately five times slower than those of WT MCF-7 cells. In mice implanted with tamoxifen pellets, tumors of PC1- or furin-transfected MCF-7 cells regressed approximately five times slower than the WT tumors. This study shows that the over-expression of proprotein convertases confers a greater estrogen dependency and anti-estrogen resistance on human breast cancer cells.     

Donor immunization with WT1 peptide augments antileukemic activity after MHC-matched bone marrow transplantation

The curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part because of immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilms Tumor 1 (WT1) peptides, induces a T-cell population that is tumor antigen specific. We determined whether allogeneic BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent antitumor activity than either therapy alone. WT1 peptide vaccinations of healthy donor mice induced CD8(+) T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that peptide immunization was effective as a prophylactic vaccination before tumor challenge, yet was ineffective as a therapeutic vaccination in tumor-bearing mice. BMT from vaccinated healthy MHC-matched donors, but not syngeneic donors, into recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of FBL3 leukemia. The transfer of total CD8(+) T cells from immunized donors was more effective than the transfer of WT1-tetramer(+)CD8(+) T cells and both required CD4(+) T-cell help for maximal antitumor activity. These findings show that WT1 peptide vaccination of donor mice can dramatically enhance GvT activity after MHC-matched allogeneic BMT.     

Development of interferon-alpha resistant subline from human chronic myelogenous leukemia cell line KT-1

OBJECTIVE: Interferon-alpha (IFN-alpha) is one of the most effective therapeutic agents for a number of hematological malignancies, including chronic myelogenous leukemia (CML). Nevertheless, its efficacy is limited because of the development of resistance to IFN-alpha therapy. Previously, we established the novel human CML cell line KT-1, which is sensitive to the antiproliferative effects of IFN-alpha. Here, we report the establishment of an IFN-alpha-resistant subline, KT-1/A3R alpha 1000, by culturing KT-1/A3 cells (IFN-alpha-sensitive subline of KT-1) with increasing concentrations of IFN-alpha, in order to analyze the mechanism of acquisition of IFN-alpha resistance in CML cells after IFN-alpha therapy. SUBJECTS AND METHODS: We developed an IFN-alpha-resistant tumor cell variant, KT-1/A3R alpha 1000, from the KT-1/A3 cell line by culturing cells with increasing concentrations of IFN-alpha. This subline was examined for its ability to proliferate and its resistance to apoptosis in high concentrations of IFN-alpha. The induction of the ISGF3 complex in response to IFN-alpha alpha in KT-1/A3R alpha 1000 was compared with that in the parental cell. RESULTS: The levels of interferon-stimulated gene factor 3 components (STAT1, STAT2, and p48) proteins and STAT2 tyrosine phosphorylation induced after IFN-alpha treatment were unchanged, but formation of the ISGF3 complex was remarkably reduced in KT-1/A3R alpha 1000 cells compared to parental cells. CONCLUSION: The KT-1/A3R alpha 1000 subline is a useful model for studying the mechanism of IFN-alpha resistance after IFN-alpha therapy.     

Wilms’ Tumor Gene WT1: Its Oncogenic Function and Clinical Application

The Wilms' tumor gene WT1 is a gene responsible for the childhood renal tumor. Wilms' tumor, and is defined as a tumor suppressor gene. However, the wild-type WT1 gene is highly expressed in leukemic blast cells of myeloid and lymphoid origin, and thus, WT1 messenger RNA provides a novel tumor marker for detection of minimal residual disease of leukemias and for monitoring disease progression of myelodysplastic syndromes. The WT1 gene exerts an oncogenic function rather than a tumor-suppressor gene function in solid tumors as well as leukemias, and the WT1 gene product is an attractive tumor antigen capable of eliciting cytotoxic T lymphocytes against WT1-expressing tumors.     

Integrin alpha 11 regulates IGF2 expression in fibroblasts to enhance tumorigenicity of human non-small-cell lung cancer cells

Integrin alpha11 (ITGA11/alpha11) is localized to stromal fibroblasts and commonly overexpressed in non-small-cell lung carcinoma (NSCLC). We hypothesized that stromal alpha11 could be important for the tumorigenicity of NSCLC cells. SV40 immortalized mouse embryonic fibroblasts established from wild-type (WT) and Itga11-deficient [knockout (KO)] mice were tested for their tumorigenicity in immune-deficient mice when implanted alone or coimplanted with the A549 human lung adenocarcinoma cells. A549 coimplanted with the fibroblasts showed a markedly enhanced tumor growth rate compared with A549, WT, or KO, which alone formed only small tumors. Importantly, the growth was significantly greater for A549+WT compared with A549+KO tumors. Reexpression of human alpha11 cDNA in KO cells rescued a tumor growth rate to that comparable with the A549+WT tumors. These findings were validated in two other NSCLC cell lines, NCI-H460 and NCI-H520. Gene expression profiling indicated that IGF2 mRNA expression level was >200 times lower in A549+KO compared with A549+WT tumors. Stable short-hairpin RNA (shRNA) down-regulation of IGF2 in WT (WT(shIGF2)) fibroblasts resulted in a decreased growth rate of A549+WT(shIGF2), compared with A549+WT tumors. The results indicate that alpha11 is an important stromal factor in NSCLC and propose a paradigm for carcinoma-stromal interaction indirectly through interaction between the matrix collagen and stromal fibroblasts to stimulate cancer cell growth.     

Antiangiogenic gene therapy for hepatocellular carcinoma using angiostatin gene

Recent studies have reported that antiangiogenic gene delivery into cancer cells inhibits growth of certain tumors in vivo. Hepatocellular carcinoma (HCC) is a hypervascular cancer, and antiangiogenic gene therapy might be suitable for HCC. In the present study, we investigated the antiangiogenic effects of angiostatin gene transduction into HCC both in vitro and in vivo. Angiostatin gene was cloned into a pSecTag2B mammalian expression vector to construct pSecTag2B-ANG. pSecTag2B or pSecTag2B-ANG were transfected into an HCC cell line, PLC/PRF/5, and then stable transfectants were obtained by Zeocin selection. pSecTag2B or pSecTag2B-ANG transfection did not alter the expression of vascular endothelial growth factor (VEGF), a potent angiogenic stimulator, or pigment epithelium-derived factor (PEDF), an angiogenic inhibitor, in PLC/PRF/5 cells. However, conditioned media (CM) derived from pSecTag2B-ANG-transfected PLC/PRF/5 cells (CM-ANG) suppressed the proliferation and migration of human umbilical vein endothelial cells (HUVEC) by 35% and 50%, respectively, relative to their effects on nontransfected cells. In in vivo experiments, pSecTag2B-ANG stable transfected (CM-Mock) and nontransfected cells (CM-N) were mixed at various proportions and the mixed cells were subcutaneously implanted into athymic mice. Suppression of tumor growth was noted in mice implanted with angiostatin gene-transfected cells, and such suppression was proportional with the percentage of transfected cells. Analysis of the vascular density in these tumors showed that the tumor growth suppression effect of angiostatin gene correlated with suppression of tumor vascularity. In conclusion, antiangiogenic gene therapy using angiostatin gene is potentially suitable for the treatment of patients with HCC.     

WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells

Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies.     

Establishment and characterization of a rectal cancer model in mice: application to cytokine gene therapy

BACKGROUND AND AIMS: We established an orthotopic animal model of rectal cancer in mice and applied this model to the study of the antitumor effects of cytokine-assisted tumor vaccine. MATERIALS AND METHODS: The CT-26 murine colon adenocarcinoma cells were inoculated into the submucosa of the rectum of the mice to induce the rectal tumor. The tumor growth rate and the survival time of the mice were observed. The cDNA of granulocyte-macrophage colony-stimulating factor (GM-CSF) was transduced to the CT-26 cell line via a retroviral vector, and the therapeutic effects of irradiated GM-CSF secreting tumor vaccine on the rectal tumor were investigated. RESULTS: All the mice implanted with the wild-type tumor cells had tumor growth in the rectum and died. The mean survival time of the mice was 28.9 days. Two doses of irradiated GM-CSF secreting tumor vaccine administered on days 0 and 3 after tumor cell implantation significantly prolonged the survival of the mice with rectal tumor compared with that of the control groups ( P<0.0001). In contrast, no antitumor effect was observed when the treatment with GM-CSF secreting tumor vaccine was delayed to 3 days after tumor cell implantation ( P>0.17). CONCLUSION: The results suggest that cytokine gene therapy exerts an antitumor effect on small tumors and may be considered as an adjuvant immunotherapy of rectal cancers and prevention of reimplantation of tumor cells disseminated during or following surgery. The orthotopic animal model of the rectal cancer in mice could be applied to the in vivo experimental studies of rectal cancer.     

Interferon-alpha downregulates the abnormal intracytoplasmic free calcium concentration of tumor cells in hairy cell leukemia.

Hairy cell leukemia (HCL) is a B-cell tumor affecting the preplasma stage of B-cell differentiation. One important feature of the disease is its exquisite sensitivity to interferon-alpha (IFN-alpha) therapy. Because we showed earlier that the CD20 molecule is consistently hyperphosphorylated in hairy cells and because previous studies showed that CD20 is involved in regulating intracytoplasmic free calcium concentrations ([Ca2+]i) in normal B lymphocytes, we measured [Ca2+]i in tumor cell samples from patients with HCL and studied the effect of IFN-alpha on this parameter. Using the Ca(2+)-sensitive fluorophore fura-2, we observed that hairy cells display a slightly but consistently higher [Ca2+]i than normal 48-hour-activated B cells or other leukemic cells. Furthermore, both in vitro preincubation of cell samples with IFN-alpha and in vivo administration of this cytokine reduced the [Ca2+]i in hairy cells. This effect was observed together with a decrease in transmembrane Ca2+ influx. However, preincubation with IFN-gamma had no effect. The in vivo correlation between the diminution of CD20 phosphorylation and [Ca2+]i in tumor cell samples from patients at the beginning of IFN-alpha therapy suggests that these two parameters are connected.     

Uncoupling protein 2 deficiency of non-cancerous tissues inhibits the progression of pancreatic cancer in mice

Background: Pancreatic ductal adenocarcinoma(PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over time. How aging of non-cancerous tissues of the host affects tumor progression, however, remains largely unknown. Methods: We took advantage of a model of accelerated aging, uncoupling protein 2-deficient( Ucp2 knockout, Ucp2 KO) mice, to investigate the growth of orthotopically trans...     

Transient receptor potential channel TRPM8 agonists stimulate calcium influx and neurotensin secretion in neuroendocrine tumor cells

TRPM8 is a member of the melastatin-type transient receptor potential ion channel family. Activation by cold or by agonists (menthol, icilin) induces a transient rise in intracellular free calcium concentration ([Ca(2+)](i)). Our previous study demonstrated that Ca(2+)-permeable cation channels play a role in IGF-1-induced secretion of chromogranin A in human neuroendocrine tumor (NET) cell line BON [Mergler et al.: Neuroendocrinology 2006;82:87-102]. Here, we extend our earlier study by investigating the expression of TRPM8 and characterizing its impact on [Ca(2+)](i) and the secretion of neurotensin (NT). We identified TRPM8 expression in NET BON cells by RT-PCR, Western blotting and immunofluorescence staining. Icilin increased [Ca(2+)](i) in TRPM8-transfected human embryonic kidney cells (HEK293) but not in mock-transfected cells. Icilin and menthol induced Ca(2+) transients in BON cells as well as in primary NET cell cultures of two different pancreatic NETs as detected by single cell fluorescence imaging. Icilin increased non-selective cation channel currents in BON cells as detected by patch-clamp recordings. This activation was associated with increased NT secretion. Taken together, this study demonstrates for the first time the expression TRPM8 in NET cells and its role in regulating [Ca(2+)](i) and NT secretion. The regulation of NT secretion in NETs by TRPM8 may have a potential clinical implication in diagnosis or therapy.